GDP/GTP Exchange Factors: Nucleus Accumbens Plasticity

GDP/GTP 交换因子：伏核可塑性

• 批准号: 7050172
• 负责人: ELIZABETH ANNE EIPPER
• 金额: $ 31.86万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7050172
• 关键词: DARPP; actins; bone density; cell morphology; cocaine; cyclin dependent kinase; cytoskeleton; dendrites; electrospray ionization mass spectrometry; fluorescence recovery after photobleaching; guanine nucleotide binding protein; guanine nucleotide exchange factors; immunocytochemistry; laboratory rat; neural plasticity; neurogenesis; neuropharmacology; newborn animals; nucleus accumbens; phosphoprotein phosphatase; phosphorylation; protein kinase A; protein localization; protein protein interaction; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): Chronic abuse of substances such as cocaine leads to structural changes in the neurons of the ventral tegmental area/nucleus accumbens. The changes in dendritic branching, spine density and spine morphology that occur in the neurons comprising this common reward pathway presumably underlie some of the behavioral changes that characterize addiction. Although it is clear that DARPP-32 (Dopamine and cAMP-Regulated PhosphoProtein, 32 kDa) and PP-1 (Protein phosphatase-1) play key roles in integrating the signals impinging on medium spiny neurons, it is not yet clear how structural changes are regulated. Rho family small GTP binding proteins and the families of GDP/GTP exchange factors and GTPase activating proteins that control their activation are key regulators of cytoskeletal dynamics. Our preliminary data reveal a substantial increase in expression of Kalirin, a Rho family GDP/GTP exchange factor, following chronic cocaine treatment of adult male rats. Exogenous expression of Kalirin in organotypic slice cultures increases spine density, while antisense-mediated reductions in Kalirin levels result in a decrease in spine density. We will first complete our exploration of the effects of chronic cocaine exposure on Kalirin. Kalirin-7 interactors will be identified through immunological and biochemical analysis of extracts prepared from control and cocaine-treated rats. The major sites at which Kalirin-7 is phosphorylated under control and cocaine-treated conditions will be identified by tandem mass spectroscopic analysis of proteolytic digests. The alterations in spine-like structures observed in medium spiny neurons following changes in Kalirin-7 expression will be evaluated using biolistic transfection of organotypic slice cultures and primary neuronal cultures. Spine dynamics will be examined using time lapse imaging and GFP-tagged Kalirin. Our preliminary data demonstrate that several well-described cocaine-responsive signaling proteins interact with Kalirin. Cdk5 phosphorylates Kalirin-7 along with DARPP-32 and Pak. Kalirin activates and forms a complex with Pak, a key regulator of actin polymerization. Protein kinase A, which phosphorylates and inactivates Pak, also phosphorylates Kalirin. PP-1, a DARPP-32 target localized to spines, also binds to Kalirin. It is our hypothesis that Kalirin-7, through its interactions with these regulators, integrates the effects of diverse signaling pathways on spine formation.

描述（由申请人提供）： 慢性滥用可卡因等物质会导致腹侧段区域/伏隔核神经元的结构变化。神经元中发生的树突分支，脊柱密度和脊柱形态的变化，包括这种常见的奖励途径，大概是某些表征成瘾的行为变化的基础。尽管很明显，DARPP-32（多巴胺和cAMP调节的磷蛋白，32 kDa）和PP-1（蛋白质磷酸酶-1）在整合撞击中刺神经元上的信号方面起着关键作用，但尚不清楚结构的变化如何变化受调节。 Rho家族小型GTP结合蛋白以及控制其激活的GDP/GTP交换因子和GTPase激活蛋白的家族是细胞骨架动力学的关键调节剂。我们的初步数据表明，在慢性可卡因对成年雄性大鼠治疗后，Kalirin（Rho家族GDP/GTP交换系数）的表达大幅增加。卡利林在器官型切片培养物中的外源表达增加了脊柱密度，而反义介导的卡利林水平的降低会导致脊柱密度降低。我们将首先完成对慢性可卡因暴露对卡利林的影响的探索。 Kalirin-7相互作用者将通过对对照和可卡因处理的大鼠制备的提取物进行免疫学和生化分析来鉴定。在对照和可卡因处理的条件下磷酸化的Kalirin-7的主要部位将通过蛋白水解消化物的串联质谱分析来鉴定。在Kalirin-7表达变化后，将使用器官型切片培养物和原发性神经元培养物的生物学转染评估，在中刺神经元中观察到的脊柱状结构的改变。脊柱动力学将使用延时成像和GFP标记的卡利林进行检查。我们的初步数据表明，几种描述的可卡因反应性信号蛋白与卡利林相互作用。 CDK5与DARPP-32和PAK一起磷酸化Kalirin-7。 Kalirin激活并形成与肌动蛋白聚合的关键调节剂PAK的复合物。蛋白激酶A磷酸化并使PAK失活，也磷酸化Kalirin。 PP-1是局部与棘突的DARPP-32靶标，也与Kalirin结合。我们的假设是，Kalirin-7通过其与这些调节剂的相互作用，整合了各种信号通路对脊柱形成的影响。